This project addresses the development of primary cytolytic T lymphocytes (CTL) in the human system. CTL are antigen-specific and constitute one of several important host resistance mechanisms. They have been implicated in anti-tumor and -viral immunity and in the converse problems of graft rejection and graft-vs-host disease in allogeneic bone marrow transplantation. While many studies have examined the effector function of CTL from cell lines or polyclonal mitogen-stimulated cultures, there remain important unknowns. Dendritic cells (DCs) constitute a trace subpopulation of blood leukocytes, but they have potent stimulatory properties for primary T cell responses. Accordingly, the biology of primary CTL development will be studied in the allogeneic mixed leukocyte reaction, using dendritic cells in comparison with other candidate APCs. Specifically, how do potential APCs bind resting AD8+ T cells and sensitize CTL precursors to MHC antigens in the allogeneic MLR? Responding lymphocytes bind to active APCs in stable clusters and can be physically separated from nonresponding lymphocytes. Recent studies using bulk lymphocytes suggest that uv- irradiated DCs bind, but do not activate responding lymphocytes normally in the MLR. This provides a novel approach by which binding and activation of CTLp can be investigated for the first time as independent events. The role of exogenous cytokines with regard to CTL proliferation and differentiation will be evaluated in this system as well. Alloantigen-specific CD8+ cells can also be cloned from single cell precursors using defined APC populations and crude cytokine supernatant. The ability to clone such CTL from both unprimed single precursors, and from sensitized cluster-derived T cells, will provide a rigorous means of studying the cellular and cytokine requirements for the early responses of primary CTL. An improved understanding of primary CTL generation should help elucidate cellular mechanisms responsible for graft rejection, graft- vs-host disease, and graft-vs-leukemia effect in allogeneic bone marrow transplantation.
